Our goal is to establish a p53-independent chemotherapy for ALL leukemias based upon the intracellular generation and manipulation of the pro-death lipid, ceramide. We reported that fenretinide (4-HPR) increased de novo ceramide synthesis in cancer cell lines in vitro, and inhibitors of ceramide catabolism, such as safingol and PPMP, synergistically increased 4-HPR cytotoxicity. We have developed formulations 4-HPR and safingol for clinical testing. We hypothesize that combinations of 4-HPR, safingol, and PPMP, will have anti-leukemia activity with tolerable systemic toxicity in vivo, and that defining the mechanisms of 4-HPR synergy in ALL cell lines will facilitate clinical development. AIM 1. Define the molecular mechanisms of 4-HPR cytotoxicity. A) Determine the dependence of 4-HPR cytotoxicity and synergy upon de novo ceramide by abrogating de novo ceramide synthesis using antisense and siRNA to the LCB1 subunit of SPT. B) Determine the molecular ordering of 4-HPR cytotoxicity by measuring ceramide, ROS, mitochondrial membrane potential (psi/m), cytochrome c release, and caspase activation, in parental and mitochondrial DNA-deficient (Rho) cell derivatives. AIM 2. Determine the active metabolite of safingol and its effect on 4-HPR-induced de novo ceramide catabolism. Determine the kinetics of 3H-safingol and catabolites, and effects on ceramide, sphingosine, and sphingosine kinase. Determine if L-threo-dihydroceramides or L-threo-ceramides reproduce the effects of safingol. AIM 3. Determine the effect of inhibiting acylceramide synthase (ACS) and glucosylceramide synthase (GCS) on 4-HPR cytotoxicity. Identify a preferred stereoisomer of PPMP. ACS and GCS catabolize de novo ceramide to nontoxic products. Determine the effect on 4-HPR cytotoxicity of selectively inhibiting ACS and GCS with antisense and/or siRNA. Identify an isomer of PPMP to inhibit both ACS and GCS. AIM 4. Establish the pharmacokinetic and pharmacodynamic interactions of 4-HPR, safingol, and PPMP in an ALL cells xenograft model. Correlate fenretinide and PPMP levels assayed by HPLC with ceramide levels assayed by mass spectroscopy in xenografts. Use drug and ceramide levels, xenograft response, and animal systemic toxicity to optimize delivery of 4-HPR, safingol, and PPMP.